This invention is based upon the discovery by the inventors of the iniB, iniA and iniC genes, and the proteins encoded by these genes which are induced by a broad class of antibiotics that act by inhibiting cell wall biosynthesis, including the first line antituberculosis agents, isoniazid (INH) and ethambutol (EMB). The discovery of the iniB, iniA and iniC genes, and the proteins encoded by these genes will have important implications in the identification of drugs effective against M. tuberculosis, as well as the treatment of drug-resistant mycobacterial strains.
Many highly effective classes of antibiotics work by inhibiting microbial cell wall biosynthesis. In M. tuberculosis several antibiotics, including isoniazid and ethambutol, appear to act by this general mechanism.
EMB is a selective antimycobacterial drug recommended for clinical use in 1996 (Karlson, A. G., Am Rev Resp Dis 84, 905-906 (1961)). Today, EMB remains an important component of tuberculosis treatment programs. Unfortunately, resistance to ethambutol has been described in 2-4% of clinical isolates of M. tuberculosis in the USA and other countries, and is prevalent among isolates from patients with multidrug-resistant tuberculosis (Bloch, A B., Cauthen, G M., Onorato, I M., et al. Nationwide survey of drug-resistant tuberculosis in the United States. JAMA 271, 665-671 (1994)).
EMB targets the mycobacterial cell wall, a unique structure among prokaryotes which consists of an outer layer of mycolic acids covalently bound to peptidoglycan via the arabinogalactan (Besra, G. S. & Chatterjee, D. in Tuberculosis. Pathogenesis, protection, and control (ed Bloom, B. R.) 285-306 (ASM Press, Washington DC, 1994)). Lipoarabinomannan, another cell wall component of significant biological importance, shares with arabinogalactan the overall structure of the arabinan polymer (Chatterjee, D., et al., J. Biol Chem 266, 9652-9660 (1991)).
EMB inhibits the in vivo conversion of [.sup.14 C]glucose into cell wall arabinan (Takayama, K. & Kolburn, J. O., Antimicrob Agents Chemother 33, 143-1499. (1989)), and results in the accumulation of the lipid carrier decaprenyl-P-arabinose (Wolucka, B. A., et al., J Biol Chem 269, 23328-23335 (1994)), which suggest that the drug interferes with the transfer of arabinose to the cell wall acceptor. The synthesis of lipoarabinomannan is also inhibited in the presence of EMB (Deng, L., et al. Antimicrob Agents Chemother 39, 694-701 (1995)), (Mikusova, K., et al., Antimicrob Agents Chemother 39, 2484-2489 (1995)); again, this indicates a specific effect on arabinan biosynthesis.
Isoniazid (INH) is a front-line drug in the treatment of tuberculosis. INH is a prodrug that requires activation by the catalase-peroxidase enzyme (katG) to an active form (Zhang et al., (1992) Nature 358, 591-593). It is likely that INH acts by blocking mycolic acid biosynthesis as evidenced by the physical and biochemical changes that occur at the same time as INH toxicity (Winder and Collins, (1970) J. Gen. Microbiol. 63, 41; Davidson and Takayama, (1979) Antibicrob. Agents Chemother. 16, 104). Treatment with INH leads to the accumulation of saturated hexacosanoic acid, and has been shown to inhibit the action of several enzymes thought to be involved in mycolic acid biosynthesis including InhA (Banerjee et al., (1994) Science 263, 227-230) and kasA (Mdluli et al., (1998) Science 280, 1607-1610).
Recent reports have documented a significant increase in the global incidence of drug resistant tuberculosis. Thus, there is a need for the identification and characterization of new target genes and proteins to aid in screening for new drugs. This would require the identification of genes that participate in the biosynthesis of the mycobacterial cell wall and the identification of mutants of these genes encoding proteins that confer resistance to drugs. While it is possible that the iniB, iniA, and iniC gene products are not in themselves targets for currently available antibiotics, these proteins may act to protect M. tuberculosis and other mycobacteria from toxic effects that occur when cell wall biosynthesis is inhibited by antibiotics. Novel drugs that inhibit the iniB, iniA, and iniC proteins may therefore act synergistically with other cell wall active antibiotics and prove useful in treating tuberculosis, including drug resistant tuberculosis.
Current high throughput drug screens do not usually assay agents at high concentrations because nonspecific toxic effects are common. This strategy may miss important compounds that could be modified to have higher potency. This is a particular concern for screening compounds against M. tuberculosis because many drugs may have difficulty penetrating through its lipid laden cell wall. Thus, there is a great need for new drug screens which overcome the deficiencies of the present screens for compounds against M. tuberculosis.